Project Summary: There is a critical gap in our knowledge about the toxicity and health effects of waterpipe tobacco smoke relative to other modified tobacco products, even though its prevalence has sharply risen in recent years. The social culture of waterpipes in cafs and restaurants and the perception that waterpipe smoking is less harmful than cigarettes, along with the distribution of flavored tobacco products, continues to drive its popularity among younger adults. A recent report from the CDC showed that waterpipe use among high school students rose sharply in 2014 and was even more popular than cigarettes. To provide data that directly informs and contributes to waterpipe regulatory action, we have assembled a multidisciplinary team of investigators with expertise in the measurement of harmful and potentially harmful constituents (HPHCs) in tobacco products identified by the FDA, exposure science, tobacco related biomarkers, in vitro and in vivo respiratory toxicology, ?omics? approaches and in silico modeling, and standardized water-pipe inhalation system. We will test our hypothesis that various waterpipe products and puffing regimens will generate unique profiles of harmful constituents that will yield a continuum of toxic health effects in in vivo and in vitro models. Recently, we developed a mouse model of waterpipe smoke exposure using a puffing regimen similar to the Beirut protocol, and our preliminary evidence demonstrates that acute exposure to waterpipe smoke induces airway inflammation. Furthermore, we have quantified PM2.5, CO, particle-bound PAHs, NNK, and nicotine in 46 waterpipe venues and collected biospecimens from 170 employees in those venues, which will enable us to model mainstream and secondhand exposures and compare our model data to human biospecimens. We have established an in vitro air-liquid interface (ALI) exposure system in combination with a human 3D organotypic model for comparing products for respiratory toxicology (MucilAir). Specific aim 1: To define the constituents of mainstream and secondhand waterpipe smoke using different puffing regimens and products. Specific aim 2: To conduct comparative in vitro and in vivo exposures to different waterpipe products and assess multiple effect endpoints of toxicity and genotoxicity in lungs and in vitro airway epithelia. Specific aim 3: To identify biomarkers from the in vivo and in vitro studies (SA1 and 2) and examine human urine samples from individuals with recent mainstream/secondhand exposure to waterpipe smoke. We will use bioinformatics approaches to integrate human in vivo and in vitro data from Aims 1-3 to develop an overall hazard index.